Method of increasing catalase expression or activity

ABSTRACT

The present invention generally relates to the field of fermentation technology and microorganisms useful for such fermentations. The invention also relates to materials including nucleic acids and proteins useful for altering fermentation characteristics of microorganisms, and to microorganisms comprising such nucleic acids and/or proteins.

This application is a National Stage application of InternationalApplication No. PCT/EP2016/080167, filed Dec. 8, 2016, which claims thebenefit of European Patent Application No. 15199810.1, filed on Dec. 14,2015.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The Sequence Listing, which is a part of the present disclosure, issubmitted concurrently with the specification as a text file. The nameof the text file containing the Sequence Listing is “78056SubSeglisting.txt”, which was created on Nov. 19, 2018 and is 34,581bytes in size. The subject matter of the Sequence Listing isincorporated herein in its entirety by reference.

FIELD OF THE INVENTION

The present invention generally relates to the field of fermentationtechnology and microorganisms useful for such fermentations. Theinvention also relates to materials including nucleic acids and proteinsuseful for altering fermentation characteristics of microorganisms, andto microorganisms comprising such nucleic acids and/or proteins. Inparticular, the invention relates to materials and methods forconferring, modifying or reducing microbial stress resistance againstoxidative stress.

BACKGROUND OF THE INVENTION

The biotechnological production of substances of interest is, on anindustrial scale, generally performed by cultivating a microorganism ina liquid medium, wherein said microorganism is capable of producing saidsubstance of interest under the cultivation conditions. During suchliquid fermentation, individual microorganism cells experienceconditions that vary greatly and in a complex way overtime. In responseto such changing conditions, microorganism cells may respond by alteringgene expression, which in turn may lead to an undesirably low productionof the substance of interest. There is correspondingly a need to providemicroorganisms with improved resilience against unfavourablefermentation conditions, thus allowing for an increased production of asubstance of interest compared to comparable microorganisms.

It has thus frequently been tried to determine stress conditions duringfermentations and to modify the genetic makeup of microorganisms inorder to improve their resilience against such stress conditions.Unfortunately, analysis of fermentation of conditions experienced byindividual microorganism cells and their genetic reactions to suchconditions is notoriously difficult. Wiegand et al. (Fermentationstage-dependent adaptations of Bacillus licheniformis during enzymeproduction; Microbial Cell Factories 2013, 12:120) have tried suchanalysis. However, understanding of fermentation conditions stillremains largely incomplete.

While Wiegand et al. reported that no vegetative catalase (KatA) proteinaccumulation over time could be observed in Bacillus licheniformisduring liquid fermentation production of a subtilisin protease, theinventors have surprisingly found that increased catalase activityimproves overall fermentation characteristics e.g of B. licheniformis inthe liquid fermentation production of e.g. proteases. This was even moresurprising as, according to Wiegand et al., O2 partial pressure (pO2) isseverely reduced throughout basically all stages of such fermentation.Thus, formation of hydrogen peroxide as a major stressor was not to beexpected.

It was thus an object of the present invention to provide materials andmethods for improving fermentations. It was also an object of thepresent invention to provide materials and methods for conferring,modifying or reducing microbial stress resistance against oxidativestress.

BRIEF SUMMARY OF THE INVENTION

The present invention therefore provides promoters comprising a −10 typebox and a −35 type box separated from one another by a linker section,wherein each box consists of a respective nucleotide sequence with arespective score obtainable by assigning a value to each nucleotide ateach position according to table 1 for the −10 type box and according totable 2 for the −35 type box, and wherein the score for the −10 type boxis at least 471 and for the −35 type box is at least 159, and whereinthe linker section has a length of at least 14 nucleotides and an A/Tcontent of at least 57%.

TABLE 1 position (5′->3′ direction) A C G T 1 0 0 0 100 2 0 0 100 0 3 286 30 36 4 8 5 0 88 5 94 0 2 5 6 23 11 6 59 7 73 5 6 16 8 63 20 3 14 9 22 2 95

TABLE 2 position (5′->3′ direction) A C G T 1 2 3 0 95 2 6 3 2 89 3 2 881 9 4 63 16 3 19 5 20 58 5 17 6 53 8 11 28

The promoter of the present invention preferably is a bidirectionalpromoter.

The present invention also provides nucleic acids comprising a promoteraccording to the present invention and a prokaryotic host cellcomprising said nucleic acid.

The invention also provides a fermentation method for producing afermentation product, comprising the steps of

-   a) providing a prokaryotic host cell comprising a nucleic acid    comprising a promoter according to the present invention operably    linked to a gene coding for a fermentation product, and-   b) cultivating the prokaryotic host cell under conditions allowing    for the expression of said gene coding for the fermentation product.

The invention also provides a fermentation method for producing afermentation product, comprising the steps of

-   a) providing a prokaryotic host cell comprising a nucleic acid    comprising a promoter according to the present invention operably    linked to a gene coding for a catalase, and-   b) cultivating the prokaryotic host cell under conditions allowing    for the expression of said gene coding for the catalase.

The invention also provides fermentation methods for producing afermentation product, comprising the steps of

-   a) providing a prokaryotic host cell comprising a nucleic acid    comprising a bidirectional promoter of the present invention    operably linked to a gene coding for a fermentation product on one    strand and simultaneously operably linked to a gene coding for a    catalase on the reverse strand, and-   b) cultivating the prokaryotic host under conditions allowing for    the expression of said gene coding for the fermentation product and    said gene coding for the catalase.

The invention also provides a method of increasing catalase expressionand/or catalase activity in a prokaryotic host cell, comprising the stepof operably linking a promoter according to the present invention with agene coding for a catalase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of the wild-type KatA promoter of Bacilluslicheniformis to a preferred promoter of the present invention.

FIG. 2 shows the gene expression characteristics of KatA and KatX genesin Bacillus licheniformis with a wild-type promoter and a promoteraccording to the present invention.

FIG. 3 shows a comparison of total catalase activity in wild-typeBacillus licheniformis (labelled “WT”) and in Bacillus licheniformiswherein the promoter of the KatA gene has been replaced by the preferredpromoter according to the present invention depicted in FIG. 1 (labelled“Mutant”).

FIG. 4 shows the organization of the KatA/KatX promoters in wild-typeBacillus licheniformis.

FIG. 5 shows a sequence alignment of the promoter sequences SEQ ID NO. 1(not according to the invention) and SEQ ID NO. 56-71. Only the fullsequence of SEQ ID NO. 1 is indicated. For all other sequences, a dotdenotes an identical nucleotide as in SEQ ID NO. 1 at the correspondingposition, nucleotides differing from that of SEQ ID NO. 1 at thecorresponding position are spelled out. For example, SEQ ID NO. 71differs from SEQ ID NO. 1 by 5 nucleotides.

FIG. 6A and Fig. 6B show a sequence alignment of the promoter sequencesSEQ ID NO. 2 (not according to the invention) and SEQ ID NO 72-87. Onlythe full sequence of SEQ ID NO. 2 is indicated. For all other sequences,a dot denotes an identical nucleotide as in SEQ ID NO. 2 at thecorresponding position, nucleotides differing from that of SEQ ID NO. 2at the corresponding position are spelled out. For example, SEQ ID NO.87 differs from SEQ ID NO. 2 by 5 nucleotides.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is particularly concerned with a promoter for aprokaryotic host cell as described hereinafter. The promoter of thepresent invention comprises a −10 type box and a −35 type box separatedfrom one another by a linker section. Such general promoter structure iscommon for prokaryotic cells and has been analysed for example by Jarmeret al, Microbiology 2001, 2417-2424.

By way of example, the inventors have now surprisingly found thatexpression of a gene operably linked to the promoter of the Bacilluslicheniformis KatA gene can be significantly increased by altering thelength of the linker section linking the −10 type box and the −35 typebox of said promoter. This was particularly surprising as the promoterin Bacillus licheniformis overlaps with the promoter directing theexpression of the KatX gene on the reverse (“minus”) strand, as outlinedin FIG. 4 . Thus, any alteration of the KatA promoter nucleotidesequence entailed the risk of reducing or abolishing the expression ofsaid KatX catalase, thereby potentially reducing overall catalaseexpression in Bacillus licheniformis, which would have an undesirableimpact on the fermentation characteristics and particularly the yield ofdesired fermentation products normally obtainable by use of saidBacillus in a fermentation.

The promoter of the present invention generally belongs to the class ofthe sigma-A dependent promoters. As Jarmer et al describe, suchpromoters comprise two more or less conserved nucleotide sequencemotifs, also called “boxes”, required for initiating transcription byRNA polymerase. Even though bacterial genomes and for example the genomeof Bacillus subtilis have been completely and repeatedly sequenced, thenumber of genes in such bacteria and correspondingly the number ofpromoters remains elusive. It is also generally not possible to reliablypredict if a nucleotide sequence will be functional as a sigma-A typepromoter in a given host cell. The invention provides guidance forovercoming or mitigating this unpredictability.

According to the present invention, the −10 type box and the −35 typebox each consist of a respective nucleotide sequence with a respectivescore. The score is obtainable by assigning a value to each nucleotideat each position in 5′ to 3′ direction without any gaps. For thenucleotide sequence of the −10 type box, the values are chosen accordingto table 1, and for the −35 type box the values are chosen according totable 2. The score for the −10 type box is at least 497 and the scorefor the −35 type box is at least 179. Thus, the promoter of the presentinvention allows for a limited flexibility in view of the respectivenucleotide sequences of the −10 type box and the −35 type box to accountfor desirable or tolerable promoter variability without generallycompromising expression strength of said promoters.

The skilled person understands that the −10 type box of the promoteraccording to the present invention is longer than a regular −10 type boxof for example Bacillus subtilis promoters. Such extended −10 type boxeshave been described for example by Helmann for Bacillus subtilispromoters (Nucleic Acids Research, 1995, 2351 to 2360). However, theauthor had established that there is no apparent correlation between theoccurrence or non-occurrence of an extended −10 type box and the lengthof the linker section. It was therefore surprising that by increasingthe length of the linker section the expression of a gene operablylinked to the promoter according to the present invention could beincreased significantly. It had to be expected that the promoter onwhich the current invention has been first implemented will not benefitfrom any alteration of the length of the linker section, as thispromoter, directing expression of the constitutive catalase of Bacilluslicheniformis, is vital for survival of this microorganism under oxygenstress conditions. Thus, it had to be expected that such promoter hadbeen optimised by evolution to its maximum expression strength. Contraryto these fears, the inventors have shown that even the promoterdirecting the expression of the KatA gene of Bacillus licheniformisbenefits from the increased length of the linker section as describedaccording to the invention.

The linker section is located adjacent to and connecting the −10 typebox and the −35 type box. According to the invention, the linker sectionhas a length of at least 14 nucleotides and an A/T content of at least73%. A linker of such length allows to increase gene expression comparedto a corresponding promoter wherein the linker section has a length ofonly 13 nucleotides.

Preferably, the nucleotide sequence of the linker section differs fromany of the sequences SEQ ID NO. 3 to 18 by insertion of one or morethymidine and/or adenosine nucleosides before any of the nucleotidepositions 1 to 5 of said respective sequences. This way, the requiredA/T content of the linker section can be maintained or even increased,facilitating the increase of gene expression from the promoter of thepresent invention. It is particularly preferred that the nucleotidesequence of the linker section differs from any of the aforementionedsequences SEQ ID NO. 3 to 18 by insertion of one or more thymidinenucleosides before any of nucleotides positions 1 to 5. Furthermore, itis preferred that the linker section does not comprise guanosine orcytidine nucleosides within the first 5 and the last 3 nucleotides ofthe linker section, counting in 5′ to 3′ direction.

Also preferably, the nucleotide sequence of the linker section differsfrom any of the sequences SEQ ID NO. 3 to 18 by insertion of one or morethymidine and/or adenosine nucleotides immediately before or immediatelyafter the last nucleotide position of said respective sequences. Thisway, the required A/T content of the linker section also can bemaintained or even increased. In cases where the promoter of the presentinvention is a bidirectional promoter as described herein comprising a−35 type box on the minus strand, insertion of the adenosine and/orthymidine nucleoside(s) at the end of the linker section (i.e. the “plusstrand” linker section) allows to increase the length of said linkersection without changing the distance of the −10 type box and −35 typebox on the minus strand.

The linker section of the promoter of the present invention preferablyhas a length of at most 18 nucleotides. For longer linker sections, theseparation between the −10 type box and the −35 type box is too large toallow for an efficient initiation of RNA transcription. Preferably, thelinker section has a length of 14 to 16 nucleotides, and even morepreferably has a length of 14 or 15 nucleotides. For such linkerlengths, separation between the −10 type box and the −35 type box isclose to optimal for initiation of RNA transcription by prokaryotic RNApolymerases of the sigma-A type. Most preferably, the linker section hasa length of 14 nucleotides.

The A/T content of the linker section preferably is at least 57%, evenmore preferably at least 69%, even more preferably at least 71%, evenmore preferably at least 76%, even more preferably at least 77%, evenmore preferably 57% to 92%, even more preferably 69% to 92%, even morepreferably 71% to 92%, even more preferably 76% to 92%, even morepreferably 77% to 92%, even more preferably 85% to 92%, even morepreferably 57% to 85%, even more preferably 69% to 85%, even morepreferably 71% to 85%, even more preferably 76% to 85%, even morepreferably 77% to 85%. Even more preferably the A/T content is 88% or,most preferably, 77%.

In a promoter according to the present invention, the nucleotidesequence of the −10 type box differs from any of the sequences SEQ IDNO. 19 to 31 by at most one nucleotide, and even more preferably ischosen from sequences SEQ ID NO. 19 to 31. Most preferably, thenucleotide sequence of the −10 type box differs by at most onenucleotide from the sequence SEQ ID NO. 19 or, even more preferably,consists of the sequence SEQ ID NO. 19. This sequence is encountered inthe wild-type promoter sequence of Bacillus licheniformis KatA gene andhas shown to be effective.

Accordingly, the score of the −10 type box preferably is at least 497,even more preferably at least 508, even more preferably at least 601,even more preferably at least 620, even more preferably at least 632,even more preferably at least 640, even more preferably 497 to 708, evenmore preferably 508 to 708, even more preferably 601 to 708, even morepreferably 620 to 708, even more preferably 632 to 708, even morepreferably 640 to 708, even more preferably 660 to 708, even morepreferably 620 to 660, even more preferably 632 to 660, even morepreferably 640 to 660. Even more preferably the score is 508 or, mostpreferably, 640.

Also, the nucleotide sequence of the −35 type box preferably differsfrom any of the sequences SEQ ID NO. 32 to 41 by at most one nucleotideand even more preferably consists of any of the sequences SEQ ID NO. 32to 41. The sequence SEQ ID NO. 32 is encountered in the Bacilluslicheniformis KatA wild-type promoter. It is thus preferred that thenucleotide sequence of the −35 type box differs from this sequence by atmost one nucleotide and preferably consists of this sequence SEQ ID NO.32.

Also, the score of the −35 type box preferably is at least 179, evenmore preferably at least 280, even more preferably at least 317, evenmore preferably at least 322, even more preferably at least 329, evenmore preferably 179 to 439, even more preferably 280 to 439, even morepreferably 317 to 439, even more preferably 322 to 439, even morepreferably 329 to 439, even more preferably 280 to 414, even morepreferably 317 to 414, even more preferably 322 to 414, even morepreferably 329 to 414, even more preferably 317 to 367, even morepreferably 322 to 367, even more preferably 329 to 367. Even morepreferably the score is 322 or, most preferably, 329.

The boundaries of promoter variability can also be expressed by the sumof scores of the −35 and −10 type box. This parameter accounts for theobservation that a low-scoring −35 type box may be compensated by ahigher scoring −10 type box and vice versa. The sum of scores preferablyis at least 826, even more preferably at least 830, even more preferablyat least 931, even more preferably at least 960, even more preferably atleast 969, even more preferably 826 to 1139, even more preferably 830 to1139, even more preferably 931 to 1139, even more preferably 960 to1139, even more preferably 969 to 1139, even more preferably 826 to1043, even more preferably 830 to 1043, even more preferably 931 to1043, even more preferably 960 to 1043, even more preferably 969 to1043, even more preferably 830 to 998, even more preferably 931 to 998,even more preferably 960 to 998, even more preferably 969 to 998, evenmore preferably 830 or, most preferably, 969. Where the sum of scoresincreases, resemblance of the promoter nucleotide sequence to that ofthe Bacillus licheniformis KatA wild-type promoter increases. Thus, theskilled person can be the more confident that the chosen promotersequence will be functional and will allow to materialise the benefitsof the present invention.

Correspondingly, the nucleotide sequence of the −10 type box ispreferably chosen from the sequences according to SEQ ID NO. 19 to 22and simultaneously the nucleotide sequence of the −35 type box ispreferably chosen from the sequences according to SEQ ID NO. 32 to 34.

In addition to the −10 type box, the linker section and the −35 type boxthe promoter of the present invention preferably comprises an upstreamsection having a length of 20 nucleotides. The upstream section islocated in 5′ direction upstream of and adjacent to the −35 type box.The A/T content of the upstream section is at least 70% and the Acontent of the upstream region is at least 35%. Such high A/T contentparticularly facilitates strong expression of a gene operably linked tothe promoter. Preferably, the A/T content of the upstream section is atleast 77%, even more preferably at least 80%, even more preferably atleast 87% and even more preferably at least 89%. Further preferably, theA/T content of the upstream region is at most 100%, less preferably theA/T content of the upstream region is at most 95% and even lesspreferably the A/T content is at most 90%. Correspondingly, the upstreamsection preferably consists of a nucleotide sequence being entirelycomposed of adenosine and thymidine nucleotides, less preferably theupstream section comprises one, 2 or 3 nucleotides that are notadenosine or thymidine. In the upstream section, nucleotides that areneither adenosine nor thymidine preferably are not adjacent to eachother. Further preferably, the nucleotide sequence of the upstreamsection preferably is such that the number of nucleotides that are notconsecutively repeated is at most 10 and at least 5, more preferably isat most 8 and at least 6. For example, the preferred upstream sectionsequence according to SEQ ID NO. 42 has 8 such isolated nucleotides atsequence position 1, 2, 5, 10, 11, 15, 19 and 20. For sequence SEQ IDNO. 55 the number would be 11 and the corresponding positions ofisolated nucleotides are 1, 2, 3, 4, 7, 12, 13, 17, 18, 19 and 20. Byrespecting these boundaries of isolated or non-repetitive nucleotides,it is secured that the upstream section nucleotide sequence will containapproximately 5 stretches of 2 or more repeating nucleotides, of whichpreferably at least 4 are stretches of repeating adenosine nucleotides.Such repetitions are favourable for the binding of RNA polymerase andthus allow for a high expression of a gene operably linked to thepromoter of the present invention. For the same reason, the A content ofthe upstream section preferably is at least 45%, even more preferably atleast 52% and even more preferably is at least 60%. For the avoidance ofdoubt, the term “A content” for the present invention means the numberof adenosine nucleotides relative to total sequence length without anygaps, that is 20 for the upstream section. Correspondingly, the term“A/T content” indicates the number of nucleotides that are eitheradenosine or thymidine expressed as percent of total sequence length.Preferred upstream section sequences are given herein as SEQ ID NO. 42to 55, wherein the upstream section sequence according to SEQ ID NO. 42is most preferred. Less preferred are such upstream sequences thatdiffer from the sequence according to SEQ ID NO. 42 by one nucleotide.Even less preferred are sequences for the upstream section that differfrom sequence SEQ ID NO. 42 by 2 nucleotides. Even less preferred areupstream sequences which differ from the sequence according to SEQ IDNO. 42 by 3 nucleotides. Particularly preferred upstream sequences arethose according to any of SEQ ID NO. 43, 44, 45, 46 and 47.

It is optional but preferred that the A/T content of a sequenceextending 46 nucleotides upstream (“lead-in section”), that is in 5′direction of the upstream section, is at least 70%. In the sequenceaccording to SEQ ID NO. 2, this 46 nucleotides sequence is located fromnucleotides 56 to 101.

A particularly preferred promoter according to the present inventiondiffers from the sequence according to SEQ ID NO. 1 by at most 15nucleotides, even more preferably at most 14 nucleotides, even morepreferably at most 13 nucleotides, even more preferably at most 12nucleotides, even more preferably at most 11 nucleotides, even morepreferably at most 10 nucleotides, even more preferably at most 9nucleotides, even more preferably at most 8 nucleotides, more preferablyby at most 7 nucleotides, even more preferably by at most 6 nucleotides,even more preferably by at most 5 nucleotides, even more preferably byat most 4 nucleotides, even more preferably by at most 3 nucleotides,even more preferably by at most 2 nucleotides and even more preferablyby one nucleotide, wherein in each case one such differing nucleotide isan insertion of an adenosine or thymidine nucleotide at any of positions70 to 85, preferably at any of positions 70 to 76 according to SEQ IDNO. 1. Most preferably, the inserted nucleotide is a thymidine such thatthe stretch of 7 consecutive thymidine nucleotides of SEQ ID NO. 1 or 2is extended to become a stretch of 8 thymidine nucleotides. An exampleof such sequence is given herein as SEQ ID NO. 56. As described belowwith regards to a bidirectional promoter, a nucleotide may also beinserted before or after the last nucleotide of the linker section. Asindicated above, the nucleotide sequence of the −10 type box, the −35type box and of the linker section can also vary, examples ofcorresponding sequences are given herein as SEQ ID NO. 57 to 71. As alsoindicated above, the sequence of the upstream section and the sequenceextending 46 nucleotides upstream of the upstream section can alsodiffer from the corresponding sections of sequences SEQ ID NO. 56 to 71.According to the invention, a promoter consisting of a nucleotidesequence according to SEQ ID NO. 56 is particularly preferred anddescribed in further detail in the examples. Equally preferred is apromoter obtainable by replacing, in the sequence according to SEQ IDNO. 2, the subsequence according to SEQ ID NO. 1 by the sequenceaccording to SEQ ID NO. 56.

The promoter of the present invention preferably is a bidirectionalpromoter. As described herein, a promoter controls, in the context of anmRNA transcription machinery, the binding of RNA polymerase to generallydouble-stranded DNA such that mRNA transcription is initiated and thesequence of a gene located downstream in 3′ direction of the promoter istranscribed into an mRNA molecule of corresponding nucleotide sequence.Due to the double-strandedness of DNA, a promoter can be located on anystrand of the DNA. According to the invention, the term “bidirectionalpromoter” is used to indicate a set of 2 promoters of overlappingnucleotide sequence, wherein one promoter controls mRNA transcription ofa gene operably linked thereto on one DNA strand (also called “plusstrand”) and the second promoter controls expression of a second geneoperably linked thereto and located on the counter strand (also called“reverse strand” or “minus strand”). Unless specifically indicatedherein, all references to a promoter, a sequence, −10 type box, −35 typebox, linker section, upstream section etc refer to the plus strand.

An example of a bidirectional promoter is the nucleic acid according toSEQ ID NO. 1 or SEQ ID NO. 2, which is the wild-type promoter of bothKatA and KatX genes of Bacillus licheniformis. In these sequences, the−35 box of the promoter on the minus strand falls into the linkersection on the plus strand as defined above. Thus, by increasing thelength of the (plus-strand) linker section in the promoter of thepresent invention, the distance between the −35 type box and −10 typebox on the reverse strand can also be increased. Due to the sensitivityof RNA polymerase for the distance between the −10 type box and −35 typebox, it had to be expected that a change in distance between these boxeswould result in a decrease of RNA transcription strength of the promoteron the minus strand. Surprisingly, however, such unwanted reduction oftranscription efficiency of the minus strand promoter is not observed orat least is not unduly strong.

The minus strand promoter of a bidirectional promoter of the presentinvention also comprises a −10 type box, a linker section and a −35 typebox. The restrictions indicated above for these elements do notnecessarily apply also to the corresponding elements of the minus strandpromoter. However, in preferred embodiments of the present invention thedefinitions and restrictions regarding the −10 type box and the −35 typebox also apply to these elements of the minus strand promoter. Thus, thescore of the −10 type box of the minus strand promoter preferably is atleast 497, even more preferably at least 508, even more preferably 497to 708, even more preferably 508 to 708, even more preferably 497 to660, even more preferably 508 to 660, and the score of the −35 type boxof the −strand promoter preferably is at least 179, even more preferablyat least 280, even more preferably at least 317, even more preferably atleast 322, even more preferably at least 329, even more preferably 179to 439, even more preferably 280 to 439, even more preferably 317 to439, even more preferably 322 to 439, even more preferably 280 to 414,even more preferably 317 to 414, even more preferably 322 to 414, evenmore preferably 317 to 367, even more preferably 322 to 367. Mostpreferably, the sum of scores of the −10 and −35 type box of the minusstrand promoter is less than the sum of scores of the −10 and −35 typebox of the plus strand promoter.

Also preferably, the −35 type box of the minus strand promoter islocated within the linker section of the plus strand promoter. This way,a compact, short length bidirectional promoter is obtained with minimalinterference between the plus strand promoter and the minus strandpromoter and accordingly with flexibility with regards to the exactsequences of the −35 type box and −10 type box of the plus and minusstrand promoter.

Preferably, the distance between the 10 type box and the −35 type box onthe minus strand is not increased by increasing the length of the (plusstrand) linker section. This can be achieved by inserting one or morenucleotides into the linker section “behind” the −35 box on the minusstrand, for example immediately between the −10 type box and the linkersection on the plus strand. This can also be achieved by deleting anumber of nucleotides in the upstream section of the plus strand equalto or greater than the number of nucleotides inserted in the linkersection.

The invention also provides a nucleic acid comprising a promoter of thepresent invention operably linked to a target gene. As indicated herein,the term “operably linked” indicates that a gene sequence is located in3′ direction of the −10 type box of the promoter such that an RNApolymerase can initiate transcription of the respective DNA strand toproduce an mRNA comprising a transcript of the respective genessequence. Typically, mRNA transcription is started in 5′ direction ofthe start codon; the −10 type box and the start codon are thus linked bya nucleic acid preferably comprising a ribosome binding site. For thepromoter of the present invention, the −10 type box described above isseparated from the start codon by a nucleic acid of preferably 20 to 50nucleotides in length. For the promoter derived from sequence SEQ ID NO.2, the start codon can be attached immediately adjacent to the lastnucleotide such that the distance between the last nucleotide of the −10type box (ending, in sequence SEQ ID NO. 2, on “ . . . caT”) and thefirst nucleotide of the start codon is 37. Such configuration isencountered in the KatA promoter of Bacillus licheniformis as describedin the examples and allows, as described in the examples, for aparticularly strong expression of the KatA gene.

The nucleic acid according to the present invention thus comprises anexpression cassette comprising the promoter of the present invention andoperably linked thereto a target gene. In cases where the promoter ofthe present invention is a bidirectional promoter, the nucleic acid ofthe present invention preferably comprises, for each strand, one geneoperably linked to the promoter of the respective strand. In suchconfiguration the promoter of the present invention is “sandwiched”between two genes, that is one gene on either strand. This configurationallows to make full use of the benefits conferred by the promoter of thepresent invention and particularly allows to have both genes expressedstrongly in an in a prokaryotic host cell.

The nucleic acid according to the present invention is a recombinantnucleic acid because it differs from the wild type KatA promoter ofBacillus licheniformis by the insertion of at least one nucleotide asdescribed herein.

The target gene preferably codes for an enzyme. The enzyme preferably isselected from the group consisting of catalase, protease, amylase,carbohydrase, lipase, cellulase, pullulanase, cutinase, pectinase,mannanase, arabinase, galactanase, xylanase, oxidase, e.g. laccase,peroxidase, isomerase, transferase, kinase, and phosphatase, whereinpreferred proteases are subtilisin proteases. Particularly preferredgenes are genes coding for catalases, and among these the catalase genesKatA and KatX of Bacillus species, even more preferably the catalasegenes coding for the KatX2 catalase of Bacillus pumilus, recorded in theUniprot database under any of the accession numbers B4AFT4_BACPU,A8FBF9_BACP2, A0A063Z4T4_BACPU, A0A0B0QA43_9BACI, M5RKX5_9BACI,K2MHE7_9BACI, W8QL66_BACPU, W6ANB4_BACPU, A0A0B4S5R6_9BACI,A0A059NBL2_9BACI, A0A0C2PYN3_BACPU and A0A081LAW9_9BACI, or coding for acatalase having at least 90% amino acid sequence identity to the KatX2catalase recorded under any of the aforementioned Uniprot accessionnumbers. For the avoidance of doubt, the respective sequences are thoserecorded in the database on 9 Nov. 2015.

The nucleic acid according to the present invention preferably is aconstruct or an expression vector such that a prokaryotic host cell canbe transformed with said construct or expression vector. Aftertransformation, the nucleic acid of the present invention may beintegrated into the host cell genome, for example by homologousrecombination. As the result of such integration, the host cell genomecomprises the target gene under the control of the promoter according tothe present invention. This way a stable expression of the target geneby the host cell can be achieved even after many generations of hostcell reproduction, preferably even in the absence of a selection markerlike an antibiotic resistance gene.

According to the invention, the nucleic acid does not have to beintegrated into the host cell genome and can instead be maintainedseparate therefrom. In such cases the nucleic acid according to thepresent invention preferably is a plasmid and comprises its own originof replication. This way, a high copy number of the nucleic acid of thepresent invention can be achieved in the host cell, thus furtherincreasing expression of the target gene or genes.

It is also possible that the nucleic acid according to the presentinvention is the genome of a prokaryotic host cell. As described above,this can be achieved by integrating a construct into the wild typegenome of the prokaryotic host cell. Preferably, integration is suchthat a wild type promoter similar to the promoter of the presentinvention is replaced by said promoter of the present invention,preferably by homologous recombination. This way, the benefit of thepresent invention and particularly the increase of expression strengthof the target gene compared to the corresponding wild-type can bemaintained in the host cell for many generations, preferably even in theabsence of a selection marker. Preferably, the promoter of the presentinvention replaces the wild type KatA promoter of Bacilluslicheniformis. As described in the examples, such replacement allows toincrease the expression of the catalase gene KatA without significantlycompromising the expression of the KatX catalase gene and beneficiallyalso without compromising the increase of KatX catalase expressionduring fermentation of Bacillus licheniformis.

The genome of a prokaryotic host cell can according to the inventionalso comprise two or more copies of the promoter of the presentinvention operably linked to corresponding target genes which may or maynot be different for each copy of the promoter-target gene combination.This allows to maintain a high expression strength of the target genesduring fermentation of the prokaryotic host cell.

The procaryotic host cell according to the present invention preferablybelongs to the taxonomic class of Bacilli, preferably to orderBacillales or Lactobacillales, more preferably to a family selected fromAlicyclobacillaceae, Bacillaceae, Listeriaceae, Paenibacillaceae,Pasteuriaceae, Planococcaceae, Sporolactobacillaceae, Staphylococcaceaeand, Thermoactinomycetaceae, and most preferably to genus Bacillus.Within this genus, the procaryotic host cell of the present inventionpreferably belongs to species Bacillus abyssalis, Bacillus acidiceler,Bacillus acidicola, Bacillus acidiproducens, Bacillus acidopullulyticus,Bacillus acidovorans, Bacillus aeolius, Bacillus aeris, Bacillus aerius,Bacillus aerophilus, Bacillus aestuarii, Bacillus aidingensis, Bacillusakibai, Bacillus alcaliinulinus, Bacillus alcalophilus, Bacillusalgicola, Bacillus alkalinitrilicus, Bacillus alkalisediminis, Bacillusalkalitelluris, Bacillus alkalitolerans, Bacillus alkalogaya, Bacillusaltitudinis, Bacillus alveayuensis, Bacillus amiliensis, Bacillusandreesenii, Bacillus aquimaris, Bacillus arbutinivorans, Bacillusaryabhattai, Bacillus asahii, Bacillus aurantiacus, Bacillusazotoformans, Bacillus badius, Bacillus baekryungensis, Bacillusbataviensis, Bacillus beijingensis, Bacillus benzoevorans, Bacillusberingensis, Bacillus berkeleyi, Bacillus beveridgei, Bacillusbogoriensis, Bacillus bombysepticus, Bacillus boroniphilus, Bacillusbutanolivorans, Bacillus canaveralius, Bacillus carboniphilus, Bacilluscasamancensis, Bacillus catenulatus, Bacillus cecembensis, Bacilluscellulosilyticus, Bacillus cereus group, Bacillus cf. pumilus SG2,Bacillus chagannorensis, Bacillus chandigarhensis, Bacilluschungangensis, Bacillus cibi, Bacillus circulans, Bacillus clausii,Bacillus coagulans, Bacillus coahuilensis, Bacillus cohnii, Bacilluscomposti, Bacillus coniferum, Bacillus daliensis, Bacillus danangensis,Bacillus decisifrondis, Bacillus decolorationis, Bacillus deramificans,Bacillus deserti, Bacillus djibelorensis, Bacillus drentensis, Bacilluseiseniae, Bacillus endophyticus, Bacillus endoradicis, Bacillusfarraginis, Bacillus fastidiosus, Bacillus ferrariarum, Bacillus firmis,Bacillus firmus, Bacillus flavocaldarius, Bacillus flexus, Bacillusforaminis, Bacillus fordii, Bacillus fortis, Bacillus fucosivorans,Bacillus fumarioli, Bacillus funiculus, Bacillus galactosidilyticus,Bacillus galliciensis, Bacillus gibsonii, Bacillus ginsenggisoli,Bacillus ginsengi, Bacillus ginsengihumi, Bacillus ginsengisoli,Bacillus gottheilii, Bacillus graminis, Bacillus granadensis, Bacillushackensackii, Bacillus halmapalus, Bacillus halochares, Bacillushalodurans, Bacillus halosaccharovorans, Bacillus hemicellulosilyticus,Bacillus hemicentroti, Bacillus herbersteinensis, Bacillus horikoshii,Bacillus homeckiae, Bacillus horti, Bacillus humi, Bacillus hunanensis,Bacillus hwajinpoensis, Bacillus idriensis, Bacillus indicus, Bacillusinfantis, Bacillus infemus, Bacillus intermedius, Bacillus iranensis,Bacillus isabeliae, Bacillus israeli, Bacillus isronensis, Bacillusjeotgali, Bacillus kochii, Bacillus koreensis, Bacillus korlensis,Bacillus kribbensis, Bacillus krulwichiae, Bacillus lehensis, Bacilluslentus, Bacillus litoralis, Bacillus locisalis, Bacillus longiquaesitum,Bacillus longisporus, Bacillus luciferensis, Bacillus luteolus, Bacillusmangrovensis, Bacillus mannanilyticus, Bacillus marcorestinctum,Bacillus marisflavi, Bacillus marmarensis, Bacillus massilioanorexius,Bacillus massiliosenegalensis, Bacillus megaterium, Bacillus meqaterium,Bacillus methanolicus, Bacillus methylotrophicus, Bacillus muralis,Bacillus murimartini, Bacillus nanhaiisediminis, Bacillus nealsonii,Bacillus neizhouensis, Bacillus nematocida, Bacillus niabensis, Bacillusniacini, Bacillus nitritophilus, Bacillus novalis, Bacillusoceanisediminis, Bacillus ohbensis, Bacillus okhensis, Bacillusokuhidensis, Bacillus oleronius, Bacillus olivae, Bacillus oryzae,Bacillus oshimensis, Bacillus pakistanensis, Bacillus panaciterrae,Bacillus patagoniensis, Bacillus persicus, Bacillus pervagus, Bacilluspichinotyi, Bacillus plakortidis, Bacillus pocheonensis, Bacilluspolyfermenticus, Bacillus polygoni, Bacillus pseudalcaliphilus, Bacilluspseudofirmus, Bacillus pseudomegaterium, Bacillus pseudomycoides,Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacilluspurgationiresistens, Bacillus qingdaonensis, Bacillus racemilacticus,Bacillus rhizosphaerae, Bacillus ruris, Bacillus safensis, Bacillussalarius, Bacillus saliphilus, Bacillus salsus, Bacillusselenatarsenatis, Bacillus senegalensis, Bacillus seohaeanensis,Bacillus shackletonii, Bacillus shandongensis, Bacillus siamensis,Bacillus similis, Bacillus simplex, Bacillus siralis, Bacillus smithii,Bacillus soli, Bacillus songklensis, Bacillus sporothermodurans,Bacillus stratosphericus, Bacillus stratosphericusi, Bacillussubterraneus, Bacillus subtilis group, Bacillus taeanensis, Bacillustequilensi, Bacillus thaonhiensis, Bacillus thermoalkalophilus, Bacillusthermoamyloliquefaciens, Bacillus thermoamylovorans, Bacillusthermocopriae, Bacillus thermolactis, Bacillus thermophilus, Bacillusthermoproteolyticus, Bacillus thermoterrestris, Bacillus thermotolerans,Bacillus thermozeamaize, Bacillus thioparans, Bacillus tianmuensis,Bacillus timonensis, Bacillus tipchiralis, Bacillus trypoxylicola,Bacillus vietnamensis, Bacillus vireti, Bacillus viscosus, Bacillusvitellinus, Bacillus wakoensis, Bacillus xiaoxiensis or Bacilluszhanjiangensis, more preferably to species Bacillus amyloliquefaciens,Bacillus brevis, Bacillus dausii, Bacillus coagulans, Bacilluslicheniformis, Bacillus pumilus or Bacillus subtilis, even morepreferably to species Bacillus subtilis, Bacillus licheniformis orBacillus pumilus and most preferably to species Bacillus licheniformis.Other preferred host cells belong to the genera Corynebacterium,Myceliophthora and Basfia, preferably to any of the speciesCorynebacterium glutamicum, Myceliophthora thermophila C1 and Basfiasucciniciproducens.

The invention also provides fermentation methods for producing afermentation product. The fermentation product preferably is a proteinor polypeptide of at least 5 amino acids length, even more preferably atleast 20 amino acids. In particular, the fermentation product can becoded for by the target gene as described above. However, thefermentation product can also be coded for by one or more genes locatedelsewhere and operably linked to respective promoters other than apromoter according to the present invention; such promoter-genecombinations can be located for example in the genome or inextra-genomic nucleic acids, for example on one or more plasmids, in afermentation host cell. From an economic perspective, the value of afermentation method according to the present invention may lie not inthe very fermentation product as described before, that is the proteinor polypeptide, but in a metabolite obtainable or obtained by action ofsaid protein or polypeptide. An example of such action is the conversionof one substance to another substance, for example the generation ofenergy equivalents (for example ATP) by decomposition of nutrients, forexample sugars, fatty acids and/or proteins. However, such metabolicprocesses depend on the prior formation of proteins or polypeptides andtypically of enzymes. It is thus warranted for the purposes of thepresent invention to call these proteins or polypeptides andparticularly these enzymes “fermentation product”.

A preferred fermentation method according to the present inventioncomprises the step of providing a prokaryotic host cell comprising anucleic acid, wherein the nucleic acid comprises a promoter according tothe present invention operably linked to a gene coding for afermentation product. Another preferred fermentation method according tothe present invention comprises the step of providing a prokaryotic hostcell comprising a nucleic acid which comprises, as a first promoter, apromoter according to the present invention operably linked to a genecoding for a catalase, and also comprising, on the same or anothernucleic acid molecule or, in case of a bidirectional promoter, on theminus strand of the nucleic acid molecule, a second promoter operablylinked to a gene coding for a fermentation product. Either preferredmethod further comprises the step of cultivating the prokaryotic hostcell under conditions allowing for the expression of said gene codingfor the fermentation product. In those cases where the prokaryotic hostcell comprises a gene coding for a catalase operably linked to apromoter according to the present invention, cultivating the prokaryotichost cell also entails the cultivation under such conditions as to allowfor the expression of said gene coding for the catalase. A fermentationmethod according to the present invention allows to make use of thebenefits conferred by the promoter of the present invention, andparticularly allows to produce during fermentation high amounts of thegene coding for the fermentation product (which can be, as describedbefore, a catalase), in particular by securing a high level ofexpression of the gene could coding for the fermentation product. Inthose cases where the fermentation method makes use of a promoteraccording to the present invention operably linked to a catalase genethe fermentation method benefits from an increase of catalase geneexpression and correspondingly of catalase activity compared to the wildtype KatA promoter of Bacillus licheniformis. Insofar, further detailsare given particularly by the accompanying examples.

As described above, the promoter of the present invention preferably isa bidirectional promoter, for example according to any of sequences SEQID NO. 72 to 87. Describing the promoter of the present invention by wayof analogy to the wild type bidirectional promoter of the KatA and KatXgenes of Bacillus licheniformis, the present invention allows tooperably linked two independent genes to the bidirectional promoter justas if the genes coding for KatA and/or KatX would be replaced. Describedin the examples, expression characteristics of the gene on the “KatAside” differs from that of a gene on the “KatX side” of the promoter:the gene on the “KatA side” can be transcribed and expressed at aconstitutively high level during fermentation, and the gene of the “KatXside” can have its transcription and expression increased duringfermentation time to reach the expression strength of the gene on the“KatA side”. Thus, by placing a gene on the minus strand relative to thesequences of the promoter of the present invention depicted herein, i.e.the “KatX side”, it is possible to have this gene expressed inincreasing strength during fermentation, thus effectively delaying theproduction of the corresponding gene product to later stages of afermentation process. This is particularly advantageous where such geneproduct is susceptible to damage is over time or where formation of thegene product would undesirably reduce the growth of corresponding hostcells during early stages of a fermentation process. Thus, the presentinvention beneficially allows to tailor gene expression according to thespecific needs of a fermentation method and also in view of constraintsimposed by the fermentation product in question.

The invention correspondingly also provides a fermentation method forproducing a fermentation product, comprising the steps of

-   a) providing a prokaryotic host cell comprising a nucleic acid    comprising a bidirectional promoter of the present invention    operably linked to a gene coding for a fermentation product on one    strand (plus strand) and simultaneously operably linked to a gene    coding for a catalase on the minus strand, and-   b) cultivating the prokaryotic host under conditions allowing for    the expression of said gene coding for the fermentation product and    said gene coding for the catalase.

This way, the fermentation process of the present invention benefitsfrom the strong and timely expression of both genes, that is the genecoding for the fermentation product and the gene coding for thecatalase. The expression of the gene coding for a catalase by thepromoter of the present invention (on the plus or minus strand)beneficially increases the prokaryotic host cells resilience againstoxidative stress and thereby aids in the production of the fermentationproduct by conferring or increasing protection against oxidation of thehost cell, any of its metabolic products and/or of the fermentationproduct.

As described above the invention also provides a method of increasingcatalase expression and/or catalase activity in a prokaryotic host cell,compared to the catalase expression and/or catalase activity,respectively, in wild type Bacillus licheniformis, comprising the stepof operably linking a promoter according to the present invention with agene coding for a catalase. For the avoidance of doubt, “increase ofexpression” biochemically means an increase of corresponding mRNAconcentration compared to the wild-type KatA promoter of Bacilluslicheniformis.

Nucleic acid sequences referred to in the context of the presentinvention are particularly:

SEQ ID NO. sequence comment SEQ ID ATTCAGCATTTATCATGATCAAATCAACGATAATAwt promoter NO. 1 AATTTTTCTTTATAATAATTATAAATAAATATTGTTTTTTTCTTGAGAAATGTTATCATTGTTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT wt promoter NO. 2ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA TGATCAAATCAACGATAATAAATTTTTCTTTATAATAATTATAAATAAATATTGTTTTTTTCTTGAGAAAT GTTATCATTGTTTTGTAATTAAAATTTACGCGAGGTGATCCTTTG SEQ ID TTTTCTTGAGAAA 77% A/T content wt linker NO. 3 SEQ IDTTTTCTTGTCAAA 77% A/T content linker; NO. 4 minus strand comprises“TTGACA” −35 type box SEQ ID TTGTTTTGAGAAA 77% A/T content linker; NO. 5minus strand comprises “TTCTCA” KatX −35 type box SEQ ID TTTCTTGAGAAAA77% A/T content linker; NO. 6 minus strand comprises “TTCTCA”KatX −35 type box SEQ ID TTAGATTGACAAA 77% A/T content linker; NO. 7minus strand comprises “TTGTCA” score 395 −35 type box SEQ IDTTGCATTGAGAAA 69% A/T content linker; NO. 8 minus strand comprises“TTCTCA” KatX −35 type box SEQ ID TTTGAGTGACAAA 69% A/T content linker;NO. 9 minus strand comprises “TTGTCA” score 395 −35 type box SEQ IDTCCAAATCAAAGA 69% A/T content linker; NO. 10 minus strand comprises“TTGATT” score 373 −35 type box, cf. SEQ ID NO. 36 SEQ ID TTGAGTGAGAAAA69% A/T content linker; NO. 11 minus strand comprises “TTCTCA”KatX −35 type box SEQ ID TTTTCTTGACAAG 69% A/T content linker; NO. 12minus strand comprises “TTGTCA” score 395 −35 type box SEQ IDTTTTGTGTAAAGG 69% A/T content linker; NO. 13 minus strand comprises“TTTACA” score 367 −35 type box, cf. SEQ ID NO. 37 SEQ ID TTTTCTTGAAAAA85% A/T content linker; NO. 14 minus strand comprises “TTTTCA”score 323 −35 type box, cf. SEQ ID NO. 38 SEQ ID TTTTCTTTACAAA85% A/T content linker; NO. 15 minus strand comprises “TTGTAA” score 357 −35 type box SEQ ID ATTTTTTGAGAAA 85% A/T content linker;NO. 16 minus strand comprises “TTCTCA” KatX −35 type box SEQ IDTTTATGAGAATAA 85% A/T content linker; NO. 17 minus strand comprises“TTCTCA” KatX −35 type box SEQ ID TTTTTTAAACAAA 92% A/T content linker;NO. 18 minus strand comprises “TTGTTT” KatA −35 type box SEQ IDTGTTATCAT 640 score KatA wt -10 type NO. 19 box SEQ ID TGTTATAAT708 score -10 type box NO. 20 SEQ ID TGTTATGAT 641 score -10 type boxNO. 21 SEQ ID TGATATCAT 632 score -10 type box NO. 22 SEQ ID TGTTATTAA558 score -10 type box NO. 23 SEQ ID TATTATAAT 608 score -10 type boxNO. 24 SEQ ID TAATATAAT 600 score -10 type box NO. 25 SEQ ID TATTAAAAT572 score -10 type box NO. 26 SEQ ID TATTATGTT 492 score -10 type boxNO. 27 SEQ ID ATTTATAAT 508 score -10 type box NO. 28 SEQ ID ATTTAAAAT472 score -10 type box NO. 29 SEQ ID TTTTATTTT 502 score -10 type boxNO. 30 SEQ ID TTTTTTTAT 462 score -10 type box NO. 31 SEQ ID TTGTTT329 score KatA wt −35 type NO. 32 box SEQ ID TTGACA439 score −35 type box NO. 33 SEQ ID TTGACT 414 score −35 type boxNO. 34 SEQ ID TTGAAA 401 score −35 type box NO. 35 SEQ ID TTGATT373 score −35 type box NO. 36 SEQ ID TTTACA 367 score −35 type boxNO. 37 SEQ ID TTTTCA 323 score −35 type box NO. 38 SEQ ID TTCTCA322 score −35 type box NO. 39 SEQ ID TTCATT 300 score −35 type boxNO. 40 SEQ ID TTCTTA 281 score −35 type box NO. 41 SEQ IDATAATAATTATAAATAAATA wt upstream box NO. 42 SEQ ID TATAATAATTATAAATAAATupstream box NO. 43 SEQ ID TTATAATAATTATAAATAAA upstream box NO. 44SEQ ID TTTATAATAATTATAAATAA upstream box NO. 45 SEQ IDCTTTATAATAATTATAAATA upstream box NO. 46 SEQ ID TCTTTATAATAATTATAAATupstream box NO. 47 SEQ ID ATAATAATTATAAATAAATC upstream box NO. 48SEQ ID TATAATAATTATAAATAAAC upstream box NO. 49 SEQ IDTTATAATAATTATAAATAAC upstream box NO. 50 SEQ ID TTTATAATAATTATAAATACupstream box NO. 51 SEQ ID CTTTATAATAATTATAAATC upstream box NO. 52SEQ ID TCTTTATAATAATTATAAAC upstream box NO. 53 SEQ IDCATAATAATTATAAATAGAC upstream box NO. 54 SEQ ID TCATAATAATTATAAATGACupstream box NO. 55 SEQ ID ATTCAGCATTTATCATGATCAAATCAACGATAATApromotor variant NO. 56 AATTTTTCTTTATAATAATTATAAATAAATATTGTTTTTTTTCTTGAGAAATGTTATCATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 57AATTTTTCTTTATAATAATTATAAATAAATATTGTT NO. 20TTTTTTCTTGAGAAATGTTATAATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 58AATTTTTCTTTATAATAATTATAAATAAATATTGAC NO. 33ATTTTTCTTGAGAAATGTTATCATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 59AATTTTTCTTTATAATAATTATAAATAAATATTGAC NO. 20 and SEQ ID NO. 33ATTTTTCTTGAGAAATGTTATAATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 60AATTTTTCTTTATAATAATTATAAATAAATATTGTT NO. 4TTTTTTCTTGTCAAATGTTATCATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 61AATTTTTCTTTATAATAATTATAAATAAATATTGTT NO. 4 and SEQ ID NO. 20TTTTTTCTTGTCAAATGTTATAATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 62AATTTTTCTTTATAATAATTATAAATAAATATTGAC NO. 4 and SEQ ID NO. 33ATTTTTCTTGTCAAATGTTATCATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 63AATTTTTCTTTATAATAATTATAAATAAATATTGAC NO. 4 and SEQ ID NO. 20ATTTTTCTTGTCAAATGTTATAATTGTTTTG and SEQ ID NO. 33 SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 64AATTTTTCTTTATAATAATTATAAATAAATATTGTT NO. 5TTTTGTTTTGAGAAATGTTATCATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 65AATTTTTCTTTATAATAATTATAAATAAATATTGTT NO. 5 and SEQ ID NO. 20TTTTGTTTTGAGAAATGTTATAATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 66AATTTTTCTTTATAATAATTATAAATAAATATTGAC NO. 5 and SEQ ID NO. 33ATTTGTTTTGAGAAATGTTATCATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 67AATTTTTCTTTATAATAATTATAAATAAATATTGAC NO. 5 and SEQ ID NO. 20ATTTGTTTTGAGAAATGTTATAATTGTTTTG and SEQ ID NO. 33 SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 68AATTTTTCTTTATAATAATTATAAATAAATATTGTT NO. 6TTTTTCTTGAGAAAATGTTATCATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 69AATTTTTCTTTATAATAATTATAAATAAATATTGTT NO. 6 and SEQ ID NO. 20TTTTTCTTGAGAAAATGTTATAATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 70AATTTTTCTTTATAATAATTATAAATAAATATTGAC NO. 6 and SEQ ID NO. 33ATTTTCTTGAGAAAATGTTATCATTGTTTTG SEQ IDATTCAGCATTTATCATGATCAAATCAACGATAATA promotor variant with SEQ ID NO. 71AATTTTTCTTTATAATAATTATAAATAAATATTGAC NO. 6 and SEQ ID NO. 20ATTTTCTTGAGAAAATGTTATAATTGTTTTG and SEQ ID NO. 33 SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional promotor NO. 72ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variantTGATCAAATCAACGATAATAAATTTTTCTTTATAA TAATTATAAATAAATATTGTTTTTTTTCTTGAGAAATGTTATCATTGTTTTGTAATTAAAATTTACGCGAG GTGATCCTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional promotor NO. 73ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variant with SEQ ID NO. 20TGATCAAATCAACGATAATAAATTTTTCTTTATAA TAATTATAAATAAATATTGTTTTTTTTCTTGAGAAATGTTATAATTGTTTTGTAATTAAAATTTACGCGAG GTGATCCTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional promotor NO. 74ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variant with SEQ ID NO. 33TGATCAAATCAACGATAATAAATTTTTCTTTATAA TAATTATAAATAAATATTGACATTTTTCTTGAGAAATGTTATCATTGTTTTGTAATTAAAATTTACGCGA GGTGATCCTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional promotor NO. 75ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variant with SEQ ID NO. 20TGATCAAATCAACGATAATAAATTTTTCTTTATAA and SEQ ID NO. 33TAATTATAAATAAATATTGACATTTTTCTTGAGAA ATGTTATAATTGTTTTGTAATTAAAATTTACGCGAGGTGATCCTTTG SEQ ID TCTTCTTCCTCCTTTATTTGTAATTAACAATAATTTbidirectional promotor NO. 76 ATCCCAATCCAGAAAAGTTATTCAGCATTTATCAvariant with SEQ ID NO. 4 TGATCAAATCAACGATAATAAATTTTTCTTTATAATAATTATAAATAAATATTGTTTTTTTTCTTGTCAAA TGTTATCATTGTTTTGTAATTAAAATTTACGCGAGGTGATCCTTTG SEQ ID TCTTCTTCCTCCTTTATTTGTAATTAACAATAATTTbidirectional promotor NO. 77 ATCCCAATCCAGAAAAGTTATTCAGCATTTATCAvariant with SEQ ID NO. 4 TGATCAAATCAACGATAATAAATTTTTCTTTATAAand SEQ ID NO. 20 TAATTATAAATAAATATTGTTTTTTTTCTTGTCAAATGTTATAATTGTTTTGTAATTAAAATTTACGCGAG GTGATCCTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional promotor NO. 78ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variant with SEQ ID NO. 4TGATCAAATCAACGATAATAAATTTTTCTTTATAA and SEQ ID NO. 33TAATTATAAATAAATATTGACATTTTTCTTGTCAA ATGTTATCATTGTTTTGTAATTAAAATTTACGCGAGGTGATCCTTTG SEQ ID TCTTCTTCCTCCTTTATTTGTAATTAACAATAATTTbidirectional promotor NO. 79 ATCCCAATCCAGAAAAGTTATTCAGCATTTATCAvariant with SEQ ID NO. 4 TGATCAAATCAACGATAATAAATTTTTCTTTATAAand SEQ ID NO. 20 and SEQ TAATTATAAATAAATATTGACATTTTTCTTGTCAA ID NO. 33ATGTTATAATTGTTTTGTAATTAAAATTTACGCGA GGTGATCCTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional promotor NO. 80ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variant with SEQ ID NO. 5TGATCAAATCAACGATAATAAATTTTTCTTTATAA TAATTATAAATAAATATTGTTTTTTGTTTTGAGAAATGTTATCATTGTTTTGTAATTAAAATTTACGCGAG GTGATCCTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional promotor NO. 81ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variant with SEQ ID NO. 5TGATCAAATCAACGATAATAAATTTTTCTTTATAA and SEQ ID NO. 20TAATTATAAATAAATATTGTTTTTTGTTTTGAGAAA TGTTATAATTGTTTTGTAATTAAAATTTACGCGAGGTGATCCTTTG SEQ ID TCTTCTTCCTCCTTTATTTGTAATTAACAATAATTTbidirectional promotor NO. 82 ATCCCAATCCAGAAAAGTTATTCAGCATTTATCAvariant with SEQ ID NO. 5 TGATCAAATCAACGATAATAAATTTTTCTTTATAAand SEQ ID NO. 33 TAATTATAAATAAATATTGACATTTGTTTTGAGAAATGTTATCATTGTTTTGTAATTAAAATTTACGCGA GGTGATCCTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional promotor NO. 83ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variant with SEQ ID NO. 5TGATCAAATCAACGATAATAAATTTTTCTTTATAA and SEQ ID NO. 20 and SEQTAATTATAAATAAATATTGACATTTGTTTTGAGAA ID NO. 33ATGTTATAATTGTTTTGTAATTAAAATTTACGCGA GGTGATCCTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional promotor NO. 84ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variant with SEQ ID NO. 6TGATCAAATCAACGATAATAAATTTTTCTTTATAA TAATTATAAATAAATATTGTTTTTTTCTTGAGAAAATGTTATCATTGTTTTGTAATTAAAATTTACGCGAG GTGATCCTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional promotor NO. 85ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variant with SEQ ID NO. 6TGATCAAATCAACGATAATAAATTTTTCTTTATAA and SEQ ID NO. 20TAATTATAAATAAATATTGTTTTTTTCTTGAGAAAA TGTTATAATTGTTTTGTAATTAAAATTTACGCGAGGTGATCCTTTG SEQ ID TCTTCTTCCTCCTTTATTTGTAATTAACAATAATTTbidirectional pronnotor NO. 86 ATCCCAATCCAGAAAAGTTATTCAGCATTTATCAvariant with SEQ ID NO. 6 TGATCAAATCAACGATAATAAATTTTTCTTTATAAand SEQ ID NO. 33 TAATTATAAATAAATATTGACATTTTCTTGAGAAAATGTTATCATTGTTTTGTAATTAAAATTTACGCGA GGTGATCCTTTG SEQ IDTCTTCTTCCTCCTTTATTTGTAATTAACAATAATTT bidirectional pronnotor NO. 87ATCCCAATCCAGAAAAGTTATTCAGCATTTATCA variant with SEQ ID NO. 6TGATCAAATCAACGATAATAAATTTTTCTTTATAA and SEQ ID NO. 20 and SEQTAATTATAAATAAATATTGACATTTTCTTGAGAAA ID NO. 33ATGTTATAATTGTTTTGTAATTAAAATTTACGCGA GGTGATCCTTTG SEQ IDTGTTTTGTAATTAAAATTTACGCGAGGTGATCCTT wt downstream box NO. 88 TG SEQ IDTTAGTTGTACTTAACTTTCACTCCTATGAGGTGAT downstream box NO. 89 CCTTTG SEQ IDTATTTTGTAATGAAATTTAACGCGAGGTGATCCT downstream box NO. 90 TTA SEQ IDTTTTTGGTGTAATTAAAATTTACGCGAGGTGATC downstream box NO. 91 CTTTG SEQ IDTGTTTTGTAATTTAAATTTACGCGAGGTGATCCTT downstream box NO. 92 TG SEQ IDTTTTTAGTGTAATTAAAATTTACGCGAGGTGATC downstream box NO. 93 CTTTG SEQ IDGTTTTGAAATTAAAATTTACGCGAGGTGATCCTT downstream box NO. 94 TG SEQ IDTTTTTTTTAATTAAAATTTACGCGAGGTGATCCTT downstream box NO. 95 TG SEQ IDTGTTTTATAATTAAAATTTACGCGAGGTGATCCTT downstream box NO. 96 TG SEQ IDTTTTTTTAATTAAAATTTACGCGAGGTGATCCTTT downstream box NO. 97 G SEQ IDGGTGTTGTAATTAAAATTTACGCGAGGTGATCCT downstream box NO. 98 TTG SEQ IDTGGATTATAATTAAAATTTAACGCGAGGTGATCC downstream box NO. 99 TTTG

The invention is hereinafter further described by way of examples; theexamples are provided for illustrative purposes only and are notintended to limit the invention or the scope of the claims.

EXAMPLES Example 1

Comparison of sequences of a wild-type Bacillus licheniformis promoterand of a preferred promoter according to the present invention FIG. 1depicts an alignment of the wild-type Bacillus licheniformis promoter(SEQ ID NO. 1) and the promoter according to SEQ ID NO. 56. Thealignment was performed according to the Needleman-Wunsch algorithm witha gap opening penalty of 16 and a gap extension penalty of 4. Thesequences differ by insertion of one thymidine in the linker section(compare also FIG. 4 ).

Example 2

Comparison of KatA and KatX catalase expression in Bacilluslicheniformis for a wild-type strain and a corresponding straincomprising the promoter according to the present invention of example 1

Fermentation Conditions for Cultivation of Bacillus licheniformisWild-Type and Mutant Strain:

The fermentation processes were conducted in a baffled stirred tankreactor (STR, Dasgip) with pH, pO2 and temperature probes in a mediumwith glucose as main carbon source and complex compounds. Theconcentration of O2 and CO2 were monitored by gas analyses. For thecultivations, the fedbatch mode was chosen with a start working volumeof 1 L. As bioreactors, 2 L glass vessels were used with 3 rushtonturbines with 6 blades.

Medium:

Medium preparation and sterilization for seed and main culture mediumtook place in the shake flask and bioreactor, respectively. PPG2000 wasused in all cultivations as antifoam agent.

The medium contained 30 g/L glucose, 120 g/L complex plant protein, 7.7g/L KH2PO4, 2.8 g/L (NH4)2SO4, 0.09 g/L Mn(II)SO4*H2O, 0.05 g/LFe(II)SO4*7H2O, 0.3 g/L CaCl2*2 H2O, 1.4 mL 7 L PPG2000, 0.025 g/LKanamycin. pH was adjusted to pH 8. The medium was sterilized in thebioreactor under stirred conditions (600 rpm) for 60 min at 123° C.

Seed Culture:

Seed culture were prepared in shake flasks with the same medium as batchcultivation during a 16 h process (39° C., pH 7.5, 200 rpm). Sampleswere taken regularly and pH, OD, sugars and organic acids (HPLC) as wellas protease activity were measured. The shake flasks were inoculatedfrom a fresh LB plate and transferred to the main culture at the end ofthe exponential phase after 16 h.

Main Culture:

For inoculation 2.7% of the starting working volume was used. The maincultivation was conducted at 39° C., with 30 g/L start glucoseconcentration and pO2 dependent stirred cascade from 350-1400 rpm and anaeration rate of 1.46 vvm.

Sampling and Total RNA Isolation:

During fermentation 10 ml samples were withdrawn and mixed in a 1:1ratio with RNAlater Solution (Life Technologies) and immediately placedon dry ice. Samples were carefully thawed on ice and cells pelleted bycentrifugation. Total RNA was isolated from cells using the peqGOLDBacterial RNA Kit (peqlab) following the manufacturers protocol. RNAconcentrations were determined measuring the absorbance at 260 nMfollowing dilution to a concentration of 100 ng/μl. RNA samples werestored at −80° C.

Reverse Transcription

Reverse Transcription was performed with 250 ng total RNA with randomhexamer oligonucleotides with the GoScript Reverse Transcription System(A5000) (Promega) according to the manufacturers protocol. The reactionis performed in an ABI Genamp PCR System 9700-Cycler.

qPCR:

Quantitative PCR (qPCR) was performed with LightCycler480 II (Roche) andthe GoTaq® qPCR Master Mix (A6001-Promega) with 20 μL PCR-reactionvolume and 10 pmol oligonucleotides each. SYBR Green was excited in theFAM/SYBR channel at 470 nm and the fluorescence detected at 510 nm.

Cycling Program   5 min 95° C. 0:10 min 95° C. 0:20 min 60° C. x45 0:20min 72° C. 0:10 min 95° C. 1:00 min 65° C . melting curve 97° C.ramprate 0.11° C./s 40° C. eternal

Data evaluation was performed with the software package Ideas 2.0 (RocheVersion 1.5.1.62). The ct values are determined by the method of ‘2ndderivate max’. The relative ct values (−dCT) of indicated target genesare calculated as follows:−dCT=−(ct(TARGET)−ct(REF))

qPCR Primers

Target gene Sequence katA_forward CCGCCTCTTGAGCGAAGA katA_reverseTGCTTCATCGAACCTACGATATTG katX_forward TCCTTGTCGCATTGCTTCAG katX_reverseCCCCGTGCGACCAAAG 16S GAGGGTTTCCGCCCTTTAGT rRNA_forward 16S rRNA_CCCAGGCGGAGTGCTTAA reverse

FIG. 2 shows the results of this example. In panel A expressioncharacteristics are given for wild-type Bacillus licheniformis and inpanel B expression characteristics are given for a recombinant Bacilluslicheniformis which differs from the wild-type strain of panel A in thatthe promoter of the KatA gene has been replaced by the promoter sequenceof the present invention depicted in FIG. 1 . Gene expression isdetermined by quantitative PCR of mRNA coding for KatA and KatXproteins, respectively. Panel B proves that the preferred promoteraccording to the present invention leads to a constitutively increasedexpression of the KatA gene without unduely reducing the expression ofthe KatX gene, and also that expression of the KatX gene is stillinduced during fermentation. Example 3: Comparison of total catalaseactivity Bacillus licheniformis for a wild-type strain and acorresponding strain comprising the promoter according to the presentinvention of example 1 1 ml samples were withdrawn from cultivationfollowing centrifugation. Cells were resuspended in assay buffer with 1mM PMSF (see below) and disrupted using Ribolyser (MP Biomedicals).After centrifugation, supernatant was recovered and used for catalaseactivity assay.

Catalase activity was assayed by the aminoantipyrine-phenol method.Herefore 10 μL of a catalase containing sample was incubated for 5 minat room temperature with 60 μL 0.86 mM H2O2 dissolved in a 166 mMPhosphate buffer at pH 7. After incubation residual H2O2 was detectedspectrophotometrically by adding 30 μL of a mixture containing 50 mMPhenol, 2.6 mM 4-Aminoantipyrine and 90 U/mL horseradish Peroxidase(Sigma 77332) at 520 nM in a microtiter plate reader. For calibration,catalase from bovine liver (Sigma C40) was used, by taking a serialdilution from 500 U/mL to 0.5 U/mL. For normalization purposes thecatalase activity was normalized on the total protein concentrationmeasured by the micro-BCA protein determination kit (Pierce).

The results of this example are shown in FIG. 3 . The figure shows thatoverall catalase activity is increased at least by a factor of 10compared to the wild-type strain, and during the first 5 h afterfermentation feedstart by a factor of 100.

The invention claimed is:
 1. A method of increasing catalase expressionor activity in a prokaryotic host cell, comprising the step ofintroducing into the host cell a nucleic acid comprising a promoteroperably linked to a gene coding for a catalase, the promoter comprisinga −10 type box and a −35 type box separated from one another by a linkersection, wherein each box consists of a respective nucleotide sequencewith a respective score obtainable by assigning a value to eachnucleotide at each position according to table 1 for the −10 type box:TABLE 1 position (5′->3′ direction) A C G T 1 0 0 0 100 2 0 0 100 0 3 286 30 36 4 8 5 0 88 5 94 0 2 5 6 23 11 6 59 7 73 5 6 16 8 63 20 3 14 9 22 2 95

and according to table 2 for the −35 type box: TABLE 2 position (5′->3′direction) A C G T 1 2 3 0 95 2 6 3 2 89 3 2 8 81 9 4 63 16 3 19 5 20 585 17 6 53 8 11 28

wherein the score for the −10 type box is at least 497 and for the −35type box is at least 179, wherein the linker section has a length of atleast 14 nucleotides and an A/T content of at least 57%, and wherein thenucleotide sequence of the linker section differs from any of SEQ IDNOS: 3 to 18 by insertion of a thymidine before any of nucleotidepositions 1 to 5 of these sequences.
 2. The method of claim 1, whereinthe linker section has a length of at most 18 nucleotides.
 3. The methodof claim 1, wherein the nucleotide sequence of the −10 type box differsfrom any of the sequences SEQ ID NO. 19 to 31 by at most 1 nucleotide.4. The method of claim 1, wherein the nucleotide sequence of the −35type box differs from any of the sequences SEQ ID NO. 32 to 41 by atmost 1 nucleotide.
 5. The method of claim 1, wherein an upstream sectionhaving a length of 20 nucleotides is located in 5′ direction upstream ofand adjacent to the −35 type box, wherein the A/T content of theupstream section is at least 70% and the A content of the upstreamregion is at least 35%.
 6. The method of claim 1, wherein the promotoris a bidirectional promoter.